Transcriptome Profile Analysis Reveals that CsTCP14 Induces Susceptibility to Foliage Diseases in Cucumber

Foliage diseases are prevalent in cucumber production and cause serious yield reduction across the world. Identifying resistance or susceptible genes under foliage-disease stress is essential for breeding resistant varieties, of which leaf-specific expressed susceptible genes are extremely important but rarely studied in crops. This study performed an in-depth mining of public transcriptome data both in different cucumber tissues and under downy mildew (DM) inoculation, and found that the expression of leaf-specific expressed transcription factor CsTCP14 was significantly increased after treatment with DM, as well as being upregulated under stress from another foliage disease, watermelon mosaic virus (WMV), in susceptible cucumbers. Furthermore, the Pearson correlation analysis identified genome-wide co-expressed defense genes with CsTCP14. A potential target CsNBS-LRR gene, Csa6M344280.1, was obtained as obviously reduced and was negatively correlated with the expression of the susceptible gene CsTCP14. Moreover, the interaction experiments of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid assay (Y1H) were successfully executed to prove that CsTCP14 could transcriptionally repress the expression of the CsNBS-LRR gene, Csa6M344280.1, which resulted in inducing susceptibility to foliage diseases in cucumber. As such, we constructed a draft model showing that the leaf-specific expressed gene CsTCP14 was negatively regulating the defense gene Csa6M344280.1 to induce susceptibility to foliage diseases in cucumber. Therefore, this study explored key susceptible genes in response to foliage diseases based on a comprehensive analysis of public transcriptome data and provided an opportunity to breed new varieties that can resist foliage diseases in cucumber, as well as in other crops.

the R domain, of which the bHLH domain is proved to be responsible for DNA binding, nuclear targeting, and protein-protein interactions mediation [33,34], and the R domain is present in seven CsTCP proteins and may participate in protein-protein interactions through developing a hydrophilic α-helix, in accordance with reference [35].
All 27 CsTCPs were classified into two classes, Class I (used as Class 1 hereinafter) and Class II. Class I comprised 12 CsTCPs (44.4%), and Class II contained 15 CsTCPs (55.6%). Moreover, Class II TCPs can be further divided into two subclasses: six CsTCPs in the CYC/TB1 subclass and nine CsTCPs in the CIN subclass ( Figures S2 and S3). This is consistent with previous reports. Class I and Class II TCP genes function in promoting plant development or inhibiting cell development, respectively, through activating a different function gene [33,[36][37][38]. For convenience, the two subclasses were renamed as Class 2 (CYC/TB1) and Class 3 (CIN) (referred to as such hereinafter). , which was the first reported, hence, its function was still unknown ( Figure S2 and S3). In addition, a phylogenetic tree based on 27 CsTCPs and 24 AtTCPs showed that they were clearly divided into three classes, and this result was consistent with the previous report in Arabidopsis (Figure 1) [39].

Identification of Leaf-Specific Expressed CsTCP Genes
The expression of CsTCP genes was obtained by a standard transcriptome analysis procedure based on a public dataset of RNA-seq in ten cucumber tissues, including root, stem, leaf, male flower, female flower, tendril, ovary, expanded ovary fertilized, and expanded ovary. (Figure 2) [40]. We found that 18 of the 25 detected CsTCPs specifically expressed in different tissues of cucumber. Among them, eight CsTCPs were highly expressed in leaves, including CsTCP5, CsTCP13, CsTCP14, CsTCP15, CsTCP16, CsTCP22, CsTCP25, and CsTCP27 ( Figure 2). Because CsTCP5, CsTCP13, CsTCP15, CsTCP16, and CsTCP22, were showing higher expression in other tissues, such as stem,

Identification of Leaf-Specific Expressed CsTCP Genes
The expression of CsTCP genes was obtained by a standard transcriptome analysis procedure based on a public dataset of RNA-seq in ten cucumber tissues, including root, stem, leaf, male flower, female flower, tendril, ovary, expanded ovary fertilized, and expanded ovary. (Figure 2) [40]. We found that 18 of the 25 detected CsTCPs specifically expressed in different tissues of cucumber. Among them, eight CsTCPs were highly expressed in leaves, including CsTCP5, CsTCP13, CsTCP14, CsTCP15, CsTCP16, CsTCP22, CsTCP25, and CsTCP27 ( Figure 2). Because CsTCP5, CsTCP13, CsTCP15, CsTCP16, and CsTCP22, were showing higher expression in other tissues, such as stem, ovary, flower, and tendril, CsTCP14, CsTCP25, and CsTCP27 were considered as leaf-specific expressed genes in cucumber, and they may play critical roles in leaf morphological changes and potential involvement in resistance or susceptibility to foliage diseases ( Figure 2 ovary, flower, and tendril, CsTCP14, CsTCP25, and CsTCP27 were considered as leaf-specific expressed genes in cucumber, and they may play critical roles in leaf morphological changes and potential involvement in resistance or susceptibility to foliage diseases ( Figure 2).

Figure 2.
Tissue-specific expression of CsTCP genes in cucumber. The CsTCP genes transcripts in nine tissues of cucumber 9930 were determined by public transcriptome data. The color scale showed that the increasing expression levels form black to red.

Responsive Analysis of CsTCP Genes under Downy Mildew Stress
In this study, a comprehensive expression pattern of CsTCP genes was analyzed based on a public transcriptome of the susceptible cucumber line Vlaspik under inoculation of downy mildew ( Figure 3) [41].
To investigate the potential functions of CsTCPs in resistance to DM, we performed CsTCPs expression model analysis after DM inoculation ( Figure 3). We identified that ten CsTCP genes of the 14 detected CsTCPs were differently expressed compared with the control, during the first day to the eighth day after inoculation of downy mildew, Pseudoperonospora cubensis in cucumber leaves ( Figure  3). The CsTCP1, CsTCP12, CsTCP14, CsTCP15, CsTCP23, and CsTCP27 were observed continuously upregulating from 2 to 8 dpi (days post inoculation), indicating that they were induced to play roles under DM stress, whereas CsTCP13 and CsTCP25 were increasingly expressed on specific days after DM treatment, 2 dpi and 2 to 3 dpi ( Figure 3). This result showed that these CsTCP genes were involved in susceptibility to downy mildew in cucumber, and CsTCP14 and CsTCP27 were important regulators in foliage diseases in DM because they were observed as leaf-specific genes in cucumber (Figures 2 and 3). Tissue-specific expression of CsTCP genes in cucumber. The CsTCP genes transcripts in nine tissues of cucumber 9930 were determined by public transcriptome data. The color scale showed that the increasing expression levels form black to red.

Responsive Analysis of CsTCP Genes under Downy Mildew Stress
In this study, a comprehensive expression pattern of CsTCP genes was analyzed based on a public transcriptome of the susceptible cucumber line Vlaspik under inoculation of downy mildew ( Figure 3) [41].
To investigate the potential functions of CsTCPs in resistance to DM, we performed CsTCPs expression model analysis after DM inoculation ( Figure 3). We identified that ten CsTCP genes of the 14 detected CsTCPs were differently expressed compared with the control, during the first day to the eighth day after inoculation of downy mildew, Pseudoperonospora cubensis in cucumber leaves ( Figure 3). The CsTCP1, CsTCP12, CsTCP14, CsTCP15, CsTCP23, and CsTCP27 were observed continuously upregulating from 2 to 8 dpi (days post inoculation), indicating that they were induced to play roles under DM stress, whereas CsTCP13 and CsTCP25 were increasingly expressed on specific days after DM treatment, 2 dpi and 2 to 3 dpi ( Figure 3). This result showed that these CsTCP genes were involved in susceptibility to downy mildew in cucumber, and CsTCP14 and CsTCP27 were important regulators in foliage diseases in DM because they were observed as leaf-specific genes in cucumber (Figures 2 and 3).

Responsive Analysis of CsTCP Genes after Treatment with Watermelon Mosaic Virus
To confirm the responsive expression of CsTCP genes under foliage disease stress, especially in CsTCP14, CsTCP25, and CsTCP27, a watermelon mosaic virus treatment was applied within a susceptible cucumber line, Europe 8, and then the genome-wide CsTCPs expression was investigated using qRT-PCR technology ( Figure 4). We found that all the CsTCPs were differentially expressed after WMV treatment, and eight CsTCPs were continuously upregulated with different expressed levels, such as CsTCP10, CsTCP11, CsTCP12, CsTCP14, CsTCP17, CsTCP19, CsTCP23, and CsTCP25. Among them, CsTCP11, CsTCP12, and CsTCP14 had the most inducible expression from the first day to the 18th day after inoculation of WMV ( Figure 4). Because CsTCP14, CsTCP25, and CsTCP27 were considered leaf-specific expressed genes, we presumed that CsTCP14 and CsTCP25 were key regulators in susceptibility to WMV in cucumber.

Responsive Analysis of CsTCP Genes after Treatment with Watermelon Mosaic Virus
To confirm the responsive expression of CsTCP genes under foliage disease stress, especially in CsTCP14, CsTCP25, and CsTCP27, a watermelon mosaic virus treatment was applied within a susceptible cucumber line, Europe 8, and then the genome-wide CsTCPs expression was investigated using qRT-PCR technology ( Figure 4). We found that all the CsTCPs were differentially expressed after WMV treatment, and eight CsTCPs were continuously upregulated with different expressed levels, such as CsTCP10, CsTCP11, CsTCP12, CsTCP14, CsTCP17, CsTCP19, CsTCP23, and CsTCP25. Among them, CsTCP11, CsTCP12, and CsTCP14 had the most inducible expression from the first day to the 18th day after inoculation of WMV ( Figure 4). Because CsTCP14, CsTCP25, and CsTCP27 were considered leaf-specific expressed genes, we presumed that CsTCP14 and CsTCP25 were key regulators in susceptibility to WMV in cucumber.

Responsive Analysis of CsTCP Genes after Treatment with Watermelon Mosaic Virus
To confirm the responsive expression of CsTCP genes under foliage disease stress, especially in CsTCP14, CsTCP25, and CsTCP27, a watermelon mosaic virus treatment was applied within a susceptible cucumber line, Europe 8, and then the genome-wide CsTCPs expression was investigated using qRT-PCR technology ( Figure 4). We found that all the CsTCPs were differentially expressed after WMV treatment, and eight CsTCPs were continuously upregulated with different expressed levels, such as CsTCP10, CsTCP11, CsTCP12, CsTCP14, CsTCP17, CsTCP19, CsTCP23, and CsTCP25. Among them, CsTCP11, CsTCP12, and CsTCP14 had the most inducible expression from the first day to the 18th day after inoculation of WMV ( Figure 4). Because CsTCP14, CsTCP25, and CsTCP27 were considered leaf-specific expressed genes, we presumed that CsTCP14 and CsTCP25 were key regulators in susceptibility to WMV in cucumber.

Identification of CsTCP14 Inducing Susceptibility to Foliage Disease
Given that CsTCP14, CsTCP25, and CsTCP27 were observed as leaf-specific expression genes in cucumbers ( Figure 2), and CsTCP14 and CsTCP27 were increasingly expressed after downy mildew stress in susceptible cucumber line Vlaspik (Figure 3), and CsTCP14 and CsTCP25 were upregulated under WMV treatment in the susceptible cucumber Europe 8 (Figure 4), CsTCP14 was both expressed specifically in leaves of cucumber and induced expression under foliage diseases DM and WMV stress in susceptible cucumbers. Therefore, the higher expression of CsTCP14 was in accordance with foliage disease expansion in susceptible cucumbers, and it was assumed to be the most potentially critical regulator in inducing susceptibility to foliage disease in cucumber. This result was consistent with the study that suggested that CsTCP14 homolog AtTCP4 played key roles both in leaf development and in plant immune response in Arabidopsis [18].

Subcellular Localization of Susceptible Gene CsTCP14
In this study, two methods were applied to the localization of the key regulator CsTCP14 involved in susceptibility to foliage disease. Firstly, an in-silico prediction of CsTCP14 was localized in the nucleus based on an online search (Table S1). Secondly, a transformation system was performed to validate the subcellular localization of CsTCP14. The pYBA1332::CsTCP14-GFP plasmid was constructed and bombarded into onion (Allium cepa) epidermal cells ( Figure 5A), and the fluorescence microscopy revealed that the CsTCP14-GFP fusion protein was localized in the nucleus and cytoplasm ( Figure 5A). This result was consistent with a previous study which confirmed that FvTCP7-GFP and FvTCP17-GFP were localized in both the nucleus and cytoplasm of Arabidopsis mesophyll protoplasts [42]. increasing the concentration of unlabeled cold probes. The shifted band appeared when the core binding site "GGNCCC" was mutated into "GGNAAA". Figure 5B shows that the Csa6M344280.1 gene was a direct target of CsTCP14, and the binding site 'GGNCCC' was essential for CsTCP14 binding to the CsNBS-LRR gene. To further confirm this transcriptional repression, a yeast one-hybrid assay (Y1H) test was performed in vivo ( Figure 5C). Only the Y1HGold-carrying PGADT7-CsTCP14 vectors and pBait-AbAi vectors could be grown in the SD/-Leu/AbA 100 medium, and the positive clones decreased gradually with an increase of dilution concentration. This result proved that pGADT7-TCP14 encoded protein specifically bound to the GGTCCC region of the CsNBS-LRR (Csa6M344280.1) promoter. Therefore, the EMSA and Y1H experiments validated that the susceptible regulator CsTCP14 could repress the defense gene Csa6M344280.1, resulting in susceptibility to foliage disease in cucumber. The EMSA experiment showed that the GST-TCP14 fusion protein was attenuated gradually by increasing the concentration of unlabeled cold-probes, while as it was reappeared after incubated with the mutated cold-probes. The band was indicated by the black arrow, and the experiment was performed in three replications. (C) The Y1H experiments showed that the positive clones were decreasing gradually when increasing the dilution concentration of pGADT7-CsTCP14. The empty vector pGADT7 was set up as negative controls, and three replications were performed.

Identification of Leaf-Specific Expressed CsTCPs in Regulating Leaf Morphogenesis
It is well known that gene expression is correlated with its function [43]. This study analyzed the expression patterns of all 27 CsTCP genes in ten different tissues of cucumber, including leaf, stem, ovary, flower, and tendril. We found that CsTCP14, CsTCP25, and CsTCP27 were observed specifically expressed in leaves ( Figure 2). As previously reported, the close genetic homologous genes of CsTCP14, CsTCP25, and CsTCP27 in Arabidopsis were AtTCP3, AtTCP4, and AtTCP24, respectively, and they were well conferred to regulate leaf development [13,15,18]. Additionally, The EMSA experiment showed that the GST-TCP14 fusion protein was attenuated gradually by increasing the concentration of unlabeled cold-probes, while as it was reappeared after incubated with the mutated cold-probes. The band was indicated by the black arrow, and the experiment was performed in three replications. (C) The Y1H experiments showed that the positive clones were decreasing gradually when increasing the dilution concentration of pGADT7-CsTCP14. The empty vector pGADT7 was set up as negative controls, and three replications were performed.

Discovery of Co-Expressed Defense Genes with the Susceptible Inducer CsTCP14
A Pearson correlation analysis between the expression of total CsTCPs and of the typical defense genes CsNBS-LRRs was conducted based on the transcriptome dataset of downy mildew inoculation, to discover the potential target genes of CsTCP14, whether they were positively or negatively involved in susceptibility to foliage disease (Table S2). The Reads Per Kilobase per Million mapped reads (RPKM) expression values of 27 CsTCPs and 48 CsNBS-LRR genes were used to calculate the correlation coefficient, nine CsNBS-LRRs were positively co-expressed with CsTCP14, and another CsNBS-LRR gene Csa6M133280.1 was significantly negatively co-expressed with CsTCP14 (Table S2). This result indicated that CsTCP14 may transcriptionally regulate these defense genes by binding with their promoters. To validate this hypothesis, the cis-acting elements of these ten CsNBS-LRR genes were further investigated, and four CsNBS-LRR genes were assumed to be target genes of CsTCPs due to harboring TCP transcription factor specific binding sites 'GGNCCC', and the significant reducing gene Csa6M344280.1 was one of the key targets, because there were three potential binding sites in its promoter (Supplementary File 1). Given that the CsTCP14 homolog AtTCP4 in Arabidopsis played an important role in transcription inhibition [33,38], CsTCP14 may target CsNBS-LRR in a transcriptional repression way, resulting in susceptibility to foliage disease in cucumber.

Repression Validation of CsTCP14 with Target Defense Gene CsNBS-LRR
In our study, an electrophoretic mobility shift assay (EMSA) test was carried out in vitro between CsTCP14 and its potential target CsNBS-LRR gene, Csa6M344280.1. The Glutathione S-transferase (GST) -tagged CsTCP14 fusion protein was confirmed to bind to the element 'GGNCCC' in the CsNBS-LRR gene promoter ( Figure 5B and Figure S4). The shifted band was attenuated gradually by increasing the concentration of unlabeled cold probes. The shifted band appeared when the core binding site "GGNCCC" was mutated into "GGNAAA". Figure 5B shows that the Csa6M344280.1 gene was a direct target of CsTCP14, and the binding site 'GGNCCC' was essential for CsTCP14 binding to the CsNBS-LRR gene. To further confirm this transcriptional repression, a yeast one-hybrid assay (Y1H) test was performed in vivo ( Figure 5C). Only the Y1HGold-carrying PGADT7-CsTCP14 vectors and pBait-AbAi vectors could be grown in the SD/-Leu/AbA 100 medium, and the positive clones decreased gradually with an increase of dilution concentration. This result proved that pGADT7-TCP14 encoded protein specifically bound to the GGTCCC region of the CsNBS-LRR (Csa6M344280.1) promoter. Therefore, the EMSA and Y1H experiments validated that the susceptible regulator CsTCP14 could repress the defense gene Csa6M344280.1, resulting in susceptibility to foliage disease in cucumber.

Identification of Leaf-Specific Expressed CsTCPs in Regulating Leaf Morphogenesis
It is well known that gene expression is correlated with its function [43]. This study analyzed the expression patterns of all 27 CsTCP genes in ten different tissues of cucumber, including leaf, stem, ovary, flower, and tendril. We found that CsTCP14, CsTCP25, and CsTCP27 were observed specifically expressed in leaves ( Figure 2). As previously reported, the close genetic homologous genes of CsTCP14, CsTCP25, and CsTCP27 in Arabidopsis were AtTCP3, AtTCP4, and AtTCP24, respectively, and they were well conferred to regulate leaf development [13,15,18]. Additionally, CsTCP14 and CsTCP21 were observed as highly expressed in tendril base and in tendril, which was recognized as an abnormal leaf in cucumber, and CsTCP21 was reported as TEN in controlling tendril formation [21]. Moreover, the AtTCP4 and AtTCP20 were proved to regulate leaf development via Jasmonic acid (JA) pathway antagonistically in Arabidopsis, and their homologous genes, CsTCP14 and CsTCP11, were assumed to be key regulators of leaf development, maybe via the JA pathway [11]. Therefore, our results identified three key leaf-specific expressed genes (CsTCP14, CsTCP25, and CsTCP27) in cucumber, and they may play critical roles in leaf morphogenesis, and be potentially involved in the response to foliage diseases in cucumber.

Identification of Susceptible CsTCP Genes under Foliage Disease Stress
The TCP TFs were reported to participate in plant resistance response through different immune processes [22][23][24][25]. This study investigated the expression of the total CsTCPs in response to foliage diseases, which was in accordance with previous results showing that the TCP genes could participate in physiological processes of plant immunity [29]. After inoculation of DM in susceptible cucumber, six CsTCPs were observed as upregulating, including CsTCP1, CsTCP12, CsTCP14, CsTCP15, CsTCP23, and CsTCP27 (Figure 3). In addition, in treatment of WMV in susceptible cucumber, eight CsTCPs were upregulated at a high level; they were CsTCP10, CsTCP11, CsTCP12, CsTCP14, CsTCP17, CsTCP19, CsTCP23, and CsTCP25 (Figure 4). Combining with the expression of CsTCP14, CsTCP25 and CsTCP27 were observed specifically expressed in leaf tissues (Figure 2), and CsTCP14 was investigated as inducible responder under foliage disease stresses, as well as in regulating leaf morphogenesis in cucumber, especially in susceptible cucumber. Given that the CsTCP14 homolog AtTCP4 was reported as a key player both in leaf development and in plant immune response in Arabidopsis [13,15,18], CsTCP14 was further identified as the most important gene to induce susceptibility to foliage diseases in cucumber.

Transcriptional Repression Regulatory of CsTCP14 and Defense Gene
In this study, the expression of 48 defense gene CsNBS-LRRs were investigated and analyzed with the key susceptible CsTCP14 in cucumber, and the CsNBS-LRR gene Csa6M133280.1 negatively co-expressed with CsTCP14 significantly (Table S2). Moreover, the promoter of Csa6M344280.1 was observed existing in three potential binding sites of 'GGNCCC', which was the key binding site of TCPs transcription factors in plants (Supplementary File 1). Given that the cucumber line tested in this study and the genome reference line 9930 were susceptible to DM and WMV, and the defense gene Csa6M344280.1 was downregulated under foliage diseases, it was considered an extremely curtailing regulator in inducing susceptibility to foliage diseases. Then, the transcriptional regulatory mechanism was applied through EMSA and Y1H tests, and the result confirmed that CsTCP14 could repress the expression of Csa6M344280.1 by binding its promoter region. As such, we constructed one possible model of CsTCP14 inducing susceptibility to foliage diseases by transcriptionally repressing the defense gene CsNBS-LRR in cucumber ( Figure 6). This result was in accordance with previous reports that the CsTCP14 homolog AtTCP4 in Arabidopsis played an important role in transcription inhibition [33,38]. Therefore, this study established a comprehensive analysis of exploring key susceptible genes in response to foliage diseases and validated the transcriptional regulation of these genes in cucumber. Our new finding would be useful for gene edition technology in breeding new varieties resistant to foliage diseases in cucumber, as well as their homologs in other crops.

Identification of Susceptible CsTCP Genes under Foliage Disease Stress
The TCP TFs were reported to participate in plant resistance response through different immune processes [22][23][24][25]. This study investigated the expression of the total CsTCPs in response to foliage diseases, which was in accordance with previous results showing that the TCP genes could participate in physiological processes of plant immunity [29]. After inoculation of DM in susceptible cucumber, six CsTCPs were observed as upregulating, including CsTCP1, CsTCP12, CsTCP14, CsTCP15, CsTCP23, and CsTCP27 (Figure 3). In addition, in treatment of WMV in susceptible cucumber, eight CsTCPs were upregulated at a high level; they were CsTCP10, CsTCP11, CsTCP12, CsTCP14, CsTCP17, CsTCP19, CsTCP23, and CsTCP25 (Figure 4). Combining with the expression of CsTCP14, CsTCP25 and CsTCP27 were observed specifically expressed in leaf tissues (Figure 2), and CsTCP14 was investigated as inducible responder under foliage disease stresses, as well as in regulating leaf morphogenesis in cucumber, especially in susceptible cucumber. Given that the CsTCP14 homolog AtTCP4 was reported as a key player both in leaf development and in plant immune response in Arabidopsis [13,15,18], CsTCP14 was further identified as the most important gene to induce susceptibility to foliage diseases in cucumber.

Transcriptional Repression Regulatory of CsTCP14 and Defense Gene
In this study, the expression of 48 defense gene CsNBS-LRRs were investigated and analyzed with the key susceptible CsTCP14 in cucumber, and the CsNBS-LRR gene Csa6M133280.1 negatively co-expressed with CsTCP14 significantly (Table S2). Moreover, the promoter of Csa6M344280.1 was observed existing in three potential binding sites of 'GGNCCC', which was the key binding site of TCPs transcription factors in plants (Supplementary File 1). Given that the cucumber line tested in this study and the genome reference line 9930 were susceptible to DM and WMV, and the defense gene Csa6M344280.1 was downregulated under foliage diseases, it was considered an extremely curtailing regulator in inducing susceptibility to foliage diseases. Then, the transcriptional regulatory mechanism was applied through EMSA and Y1H tests, and the result confirmed that CsTCP14 could repress the expression of Csa6M344280.1 by binding its promoter region. As such, we constructed one possible model of CsTCP14 inducing susceptibility to foliage diseases by transcriptionally repressing the defense gene CsNBS-LRR in cucumber ( Figure 6). This result was in accordance with previous reports that the CsTCP14 homolog AtTCP4 in Arabidopsis played an important role in transcription inhibition [33,38]. Therefore, this study established a comprehensive analysis of exploring key susceptible genes in response to foliage diseases and validated the transcriptional regulation of these genes in cucumber. Our new finding would be useful for gene edition technology in breeding new varieties resistant to foliage diseases in cucumber, as well as their homologs in other crops.
Phylogenetic analysis was conducted by Cluster W software (Version 7), which was employed to perform protein multiple sequence alignments of 27 CsTCPs. The alignment was subsequently imported into MEGA6.0 software to construct an unrooted phylogenetic tree by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates [49]. The amino acid sequences of TCPs in Arabidopsis thaliana were obtained from previous studies [35]. The TCP evolution between cucumber and Arabidopsis was studied through an unrooted phylogenetic tree, acquired from the same method above.

Transcriptome Analysis of Leaf-Specific Expressed CsTCPs in Cucumber
The tissue-specific expression patterns of CsTCPs were analyzed based on the published RNA-seq data with an accession number of SRA046916 [40]. The transcriptome profile analysis was on 10 different cucumber tissues, including root, stem, leaf, male flower, female flower, ovary, expanded ovary, expanded ovary fertilized, and tendril. The RPKM value was recalculated by a standard transcriptome analysis protocol [6]. The genome-wide expression of CsTCP genes was shown on a heatmap using HemI 1.0 software, and the higher expression was indicated by a color bar changing from black to red.

Transcriptome Analysis of CsTCPs Regulatory after Inoculation of Downy Mildew
The expression regulation of CsTCP genes responsive to downy mildew stress was retrieved from a public transcriptome dataset of RNA-seq, which was conducted for revealing the genome-wide differential expressed genes after inoculation with Pseudoperonospora cubensis (SRP009350) [41]. The susceptible cucumber line Vlaspik was treated with downy mildew on leaves, and the leaf sample was investigated at 0, 1, 2, 3, 6, and 8 days after inoculation (dpi). Because the transcriptome was sequenced by single-end technology, the RPKM value was recalculated by a standard transcriptome analysis protocol according to a previous report [6], and the expression of CsTCP genes was shown by a heatmap using HemI 1.0 software in this study.

Materials and WMV Inoculation
The cucumber line Europe 8, which is susceptible to WMV, were cultivated in a greenhouse (Beijing Vegetable Research Center). The WMV was diluted to a concentration of 1:3 (w/v) in 0.2 mol·L −1 phosphoric acid buffer (pH 7.0) when the second true leaf was fully expanded. The leaves were inoculated with a small amount of 600 to 800 mesh emery after rinsing with clear water, and a control setting was inoculated with phosphoric acid buffer (three duplications and 20 seedlings for each treatment). Both the inoculated and control seedlings were cultivated under the correct conditions (insect-free,~30 • C, 90% relative humidity).

RNA Extraction
Leaf samples were collected from 3 independent plants in each treatment of the 0, 1, 3, 6, 9, 12, 18, and 24 days post-inoculation (dpi), and they were immediately frozen in liquid nitrogen. Total RNA was isolated with Quick RNA isolation Kit (Huayueyang, Beijing, China) following the manufacturer's recommended protocol. An aliquot of total RNA was treated with RNase-free DNase I (NEB, Ipswich, MA, USA) to degrade contaminating genomic DNA. The qualities and quantities of total RNA were assessed on NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The total RNA was subsequently reverse transcribed to double-stranded cDNA using Super Script III Reverse Transcriptase (TaKaRa, Dalian, China).

Real-Time Quantitative PCR
The specific primer design of each CsTCP gene was implemented on an online Primer3 website (http: //primer3.ut.ee), and are shown in Table S3. The reference gene sequences that quantitative real-time (qRT-PCR) used are 5 -GGCAGTGGTGGTGAACATG-3 and 5 -TTCTGGTGATGGTGTGAGTC-3 . The qRT-PCR setting to three replications was performed using an SYBR Premix Ex TaqTM kit (TaKaRa, Dalian, China) and a Roche LightCycler480 System (Bio-Rad, Hercules, CA, USA). Relative expression levels of CsTCPs were calculated with the comparative threshold method (2 −∆∆Ct ) and shown on a heatmap using HemI 1.0 software. The expression of CsTCPs under WMV stress was analyzed by real-time quantitative PCR (qRT-PCR) technology; treatments covered 1, 3, 6, 9, 12, and 18 days after inoculation.

Identification of Potential Target Genes of CsTCPs' Susceptibility to Foliage Disease
The TCP genes were reported as being involved in regulating plant immune response by transcriptional regulation of their target genes [24]. In this study, the potential target genes of CsTCPs involved in resistance or susceptibility to foliage disease were explored. A Pearson correlation analysis was conducted to investigate the correlation between the key CsTCPs and their potential targets. This study focused on the typical defense genes, CsNBS-LRRs, in cucumber, which was a key participator in response to disease. The transcriptome profile dataset under downy mildew was used, and the correlation coefficient and significance were calculated in the R psych packages, the significance correlation (p < 0.05) between CsTCP genes and CsNBS-LRRs was indicated by a star (*), and extreme significance correlation (p < 0.01) was indicated by two stars (**) in this result.

Subcellular Localization of CsTCP14
The CsTCP14 (Csa4M628330.1) coding sequence was amplified with primers listed in Table S4. Subsequently, CsTCP14 was inserted into the pYBA1332-GFP vector. Both the pYBA1332::GFP and pYBA1332::CsTCP14-GFP vectors were bombarded into onion bulb scale epidermal cells. After incubation in the dark at 25 • C for 6 h, fluorescence signals were detected using a Laser Scanning Confocal Microscope (Nikon A1R, Tokyo, Japan).

Expression of CsTCP14 Protein
The sequence of the CsTCP14 gene was amplified from cucumber 9930 with specific primers (Table S5) and cloned into the pGEX4T-1 vector carrying GST tag. The pGEX4T-1-CsTCP14 was transformed into Escherichia coli strain BL21 (DE3) for further culture. The 0.2 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the bacteria culture when the concentration reached 0.6 at 600 nm (OD600). The recombinant GST-CsTCP14 protein was harvested after culture at 37 • C for 5 h with 200 rpm. To confirm the correctness of the CsTCP14 expression, Western blot was performed to identify the fusion protein with the right Odyssey CLx Imaging System (LI-COR, Lincoln, NE, USA). The GST-CsTCP protein was purified by Bio-Scale Mini Profinity GST cartridge (Bio-Rad, CA, USA) and size exclusion column (Bio-Rad, Hercules, CA, USA).

Electrophoretic Mobility Shift Assay
The purified protein was subjected to a Light Shift Chemiluminescent EMSA kit (Thermo, Waltham, MA, USA). The CsNBS-LRR gene CSa6M344280.1 probe and its mutation probes (Table S6) were selected to identify the authenticity of the binding site. Among them, the CsNBS-LRR probes without mutation were 5 -biotin-labeled. Meanwhile, the same sequences with non-labeled biotin were used as the competitive probes for EMSA. The 20 µL binding reactions were electrophoresed in 6.5% native polyacrylamide gel, and then electro transferred onto a nylon membrane. The membrane was detected using a ChemiDocTM MP Imaging System (Bio-Rad, CA, USA).

Yeast One-Hybrid Assay
Yeast one-hybrid assay was conducted to validate the transcriptional regulation of CsTCP14 and its targets based on the protocol in Clontech's Matchmaker Gold Yeast One-Hybrid System (Clontech Laboratories, Inc., a Takara Bio Company, Mountain View, CA, USA). The pGADT7 prey vector carrying CsTCP14 was constructed using the primers pGADT7-F and pGADT7-R (Table S7). The CsNBS-LRR promoter fragment containing the TCP binding site "GGNCCC" was shown as the bait sequence. To obtain genuine positive clones, we also mutated the TCP binding site "GGNCCC" to "GGTAAA" (designed as Bait-m). The Bait and Bait-m sequences were cloned into the pAbAi vector by using the primers listed in Table S7. The linearized pBait-AbAi and pBait-m-AbAi plasmids with BstB I were cloned into yeast strain Y1HGold. The transformed yeast cells carrying an empty pGADT7 vector were set up as negative controls. The Y1HGold were cultured in the optimal concentration of AbA. The experiments were repeated three times.

Statistical Analysis
A one-way analysis of variance (ANOVA) test was employed to analyze the variance and significant difference among CsTCP expressions under different conditions. The software used for the statistical analyses was SPSS 16.0 (SPSS Company, Chicago, IL, USA).

Conclusions
In this study, we identified a key regulatory gene, CsTCP14, which was responsive to susceptibility to foliage disease, downy mildew, and watermelon mosaic virus in cucumber. The CsTCP14 was observed as a leaf-specific expressed gene and was inducible to upregulation under downy mildew and watermelon mosaic virus treatment. Further, the potential target defense gene, Csa6M344280.1, of CsTCP14 under foliage disease stress was explored and validated by EMSA and Y1H experiment. This study constructed a draft model to demonstrate that the leaf-specific expressed gene CsTCP14 was negatively regulating the defense gene Csa6M344280.1 to induce susceptibility to foliage diseases in cucumber.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/10/2582/ s1. Figure S1: Chromosome localization of CsTCP genes in cucumber. A Circos diagram illustrates the physical locations of CsTCP genes. CsTCP genes with the same color represent the same class: Class 1 in green, Class 2 in red, and Class 3 in blue. Segmental duplicated genes (CsTCP2/CsTCP6, CsTCP11/CsTCP23, and CsTCP24/CsTCP26) are indicated as curves. Figure S2: Phylogenetic tree and gene structures of CsTCP genes. The branch with different colors in the phylogenetic tree represents a different class: Class 1 in green, Class 2 in red, Class 3 in blue. The exon and intron arrangement of CsTCP genes is shown with green bars and black lines, respectively. The scale of the exon and intron are drawn at the bottom. Figure